A symbiotic footprint in the plant root microbiome

Background A major aim in plant microbiome research is determining the drivers of plant-associated microbial communities. While soil characteristics and host plant identity present key drivers of root microbiome composition, it is still unresolved whether the presence or absence of important plant root symbionts also determines overall microbiome composition. Arbuscular mycorrhizal fungi (AMF) and N-fixing rhizobia bacteria are widespread, beneficial root symbionts that significantly enhance plant nutrition, plant health, and root structure. Thus, we hypothesized that symbiont types define the root microbiome structure. Results We grew 17 plant species from five families differing in their symbiotic associations (no symbioses, AMF only, rhizobia only, or AMF and rhizobia) in a greenhouse and used bacterial and fungal amplicon sequencing to characterize their root microbiomes. Although plant phylogeny and species identity were the most important factors determining root microbiome composition, we discovered that the type of symbioses also presented a significant driver of diversity and community composition. We found consistent responses of bacterial phyla, including members of the Acidobacteria, Chlamydiae, Firmicutes, and Verrucomicrobia, to the presence or absence of AMF and rhizobia and identified communities of OTUs specifically enriched in the different symbiotic groups. A total of 80, 75 and 57 bacterial OTUs were specific for plant species without symbiosis, plant species forming associations with AMF or plant species associating with both AMF and rhizobia, respectively. Similarly, 9, 14 and 4 fungal OTUs were specific for these plant symbiont groups. Importantly, these generic symbiosis footprints in microbial community composition were also apparent in absence of the primary symbionts. Conclusion Our results reveal that symbiotic associations of the host plant leaves an imprint on the wider root microbiome – which we term the symbiotype. These findings suggest the existence of a fundamental assembly principle of root microbiomes, dependent on the symbiotic associations of the host plant. Supplementary Information The online version contains supplementary material available at 10.1186/s40793-023-00521-w.


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Figures S1 to S7 12 Tables S1 to S8 magnified intersection method with one hundred intersections per sample [2]. For legume species same Picogreen assay described above and pooled in equal amounts (15ng/sample for 16S, 30ng/sample for ITS) to obtain a 16S and an ITS library. Subsequently, the volume of each library was reduced to Both libraries were then purified and concentrated with the Agencourt AMPure XP kit (Beckman assay on a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA). The libraries were then combined 74 and concentrated once more with AMPure and eluted twice in 75µL sterile water for MiSeq library 75 preparation.

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The MiSeq libraries were prepared at the Functional Genomics Center Zurich (www.fgcz.ch) 77 with the NEBNext DNA library Ultra kit (New England Biolabs, Ipswich, MA, USA). After end-78 repairing and polyadenylating the amplicons, NEBNext Adaptors were ligated. The ligated samples 79 were run on a 2% agarose gel and the desired fragment length was excised (50 bp ± the target fragment 80 length). DNA from the gel was purified with MinElute Gel Extraction Kit (Qiagen, Hilden, Germany).

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Fragments containing NEBNext adapters on both ends were selectively enriched with PCR using four

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To identify the primary symbionts (Rhizobia and AMF) in the root microbiomes, we marked raw reads across all nodule samples of legume plants as nodule-specific symbiotic OTUs (52 OTUs).

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Likewise, ITS-OTUs annotated as Glomeromycota in root samples of all plant species were marked as 119 specific AMF OTUs (127 OTUs). We refer to these nodule-and AMF-specific OTUs collectively as 120 'primary symbionts', and for specific analyses -i.e., to test whether changes in the root microbiome S1,

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We assessed the impact of the symbiotic groups on community structure with a permutational 135 multivariate ANOVA (PERMANOVA) in R with the function adonis from the R package vegan [17] 136 with 999 permutations. This was performed on Bray-Curtis dissimilarities calculated from rarefied 137 datasets with and without the primary symbionts. For assessing the effects of the symbiotic groups on 138 both the diversity indices and community dissimilarities, we used the same model detailed below.

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The structure of all (PERM)ANOVA models followed general design principles (see Schmid 142 et al., [18] for a detailed discussion of this approach). For all models, factors were fitted sequentially 143 (type I sum of squares). Significance tests were based on F-tests calculated manually using appropriate 144 error terms and denominator degrees of freedom. To correct for differences in sequencing depth and to 145 account for differences between the two different pot types used in the experiment, the factors 146 sequencing depth and pot were fitted first. Because the symbiotic group characterized by no symbiosis 147 with AMF but with rhizobia consisted of Lupin only (R), we next fitted a factor for the plant species 148 being lupin or not (isLupin). We then fitted a factor symbiotic group, representing the symbiotic type 149 of the remaining plant species, and consisting of the levels N (No symbiosis with AMF or rhizobia), A 150 (symbiosis with AMF, but not with rhizobia), and AR (symbiosis with AMF and Rhizobia). To compare 151 the groups to each other, the factor symbiotic group was split into three different sets of contrasts. In 152 each set, one group was first compared to the others, and the remaining two groups were then compared to each other, so that the model was run three times to obtain the results for all three pairwise N (neither symbiont; Amaranthaceae and Brassicaceae) could be included as group R and AR did not 157 contain multiple families. Finally, we included three additional terms to test for differences between the 158 individual plant species that comprise symbiotype groups A, AR, and N. We chose this modeling 159 technique over other possible methods (e.g., phylogenetic least squares regression) because our  (Table S4). However, differences between 192 families in groups A and N and between plant species from groups A, AR, and N was the strongest 193 determinant of b-OTU community composition, explaining a total of 6.4% and 23.5% of overall 194 variance, respectively (Table S4). In the f-OTU community, significant differences between families in 195 groups A and N and species in groups A and AR explained a combined 15% and 16.8% of variance 196 respectively.

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In short, plant family and species identity are strong and significant drivers of differences in 198 root microbiome composition. However, as expected, the presence of rhizobia and AMF also presents 199 a significant driver community composition.

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However, cluster root structures were removed from Lupin replicates. The Genbank accession number of the rbcL gene for each species was used to produce 284 the phylogenetic tree in Figure 1.